Cellomics arrayscan vt1 hcs automated reader

After being infected with the shCtrl lentivirus or shHOXD3 lentivirus, RKO cells were seeded in 96-well plates at a concentration of 2,000 cells/well and incubated for 5 days at 37°C with 5% CO₂. The cells were counted each day using the Cellomics ArrayScan™ VT1 HCS automated reader (Cellomics Inc., Pittsburgh, PA, USA). At least 800 cells/well were analyzed. At 1, 2, 3, 4, or 5 days post-infection, the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml; Promega, Shanghai, China) at a final concentration of 0.5 mg/ml for 4 h. After discarding the supernatants, 150 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., LLC, St. Louis, MO, USA) was added to each well. The plates were read at 490 nm using an ELISA reader (Tecan Infinite, Männedorf, Switzerland). All experiments were performed in triplicate.